NMR studies of bacterial needle and tip proteins

描述（由申请方提供）：拟定NMR研究的目的是确定细菌纳米注射器组装中涉及的蛋白质-蛋白质相互作用，这在许多细菌病原体的发病机制中至关重要。纳米注射器是一种针状大分子组装体，形成了细菌蛋白质输出机制III型分泌系统（T3 SS）的结构部分。T3 SS还由注入宿主细胞质的效应蛋白和在细菌细胞质内与T3 SS蛋白形成复合物的伴侣蛋白组成。T3 SS针装置由超过20种不同的蛋白质组装而成，由锚定在细菌膜上的基底结构、细菌表面上的针本身、尖端复合物和易位子组成。易位子是跨膜结构，其在宿主细胞膜上穿孔以允许细菌蛋白质通过进入宿主细胞质。在第一轮融资中，我们已经确定了针蛋白如何与尖端蛋白相互作用。目前，tip蛋白如何与易位子相互作用是完全未知的。对于这种更新，我们的目标#1是确定尖端蛋白如何与易位子蛋白相互作用。另一个未知的是尖端蛋白如何与它们的伴侣蛋白相互作用，因此我们的目标#2是确定尖端蛋白如何与它们的伴侣蛋白相互作用。为了促进我们对T3 SS生物学的理解，我们的NMR研究将与电子显微镜和功能测定相结合。许多病原体依靠III型分泌系统感染全世界数百万人。因为针装置暴露在细菌表面上，所以破坏针组件是开发新型抗感染药物的有吸引力的目标。这种方法需要详细了解针头组装中涉及的蛋白质-蛋白质相互作用。

DESCRIPTION (provided by applicant): The objective of the proposed NMR studies is to determine the protein-protein interactions involved in the assembly of a bacterial nanoinjector that is essential in the pathogenesis of many bacterial pathogens. The nanoinjector is a needle-like macromolecular assembly and forms the structural part of bacterial protein export machinery, the type III secretion system (T3SS). The T3SS also consists of effector proteins, which are injected into the host cell cytoplasm, and chaperones, which form complexes with T3SS proteins while inside the bacterial cytoplasm. The T3SS needle apparatus is assembled from over 20 different proteins and consists of a basal structure anchored on the bacterial membranes, the needle itself on the bacterial surface, a tip complex, and a translocon. The translocon is the membrane spanning structure that punctures a hole on the host cell membrane to allow the passage of bacterial proteins into the host cell cytoplasm. On the first round of funding, we have determined how the needle protein interacts with the tip protein. Currently, how the tip protein interacts with the translocon is completely unknown. For this renewal, our Aim #1 is to determine how the tip protein interacts with the translocon proteins. Another unknown is how the tip proteins interact with their chaperones, thus our Aim #2 is to determine how the tip proteins interact with their chaperones. To advance our understanding of the biology of the T3SS, our NMR studies will be complemented with electron microscopy and functional assays. Many pathogens rely on the type III secretion system to infect millions of people worldwide. Because the needle apparatus is exposed on the bacterial surface, disrupting the needle assembly is an attractive target for the development of novel anti- infectives. This approach requires a detailed understanding of the protein-protein interactions involved in needle assembly.